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Abstract

Abstract The angiotensin II type-1 (AT1) receptor, a G protein–coupled receptor, lacks intrinsic kinase activity. However, recent data show that angiotensin II (Ang II) stimulates tyrosine phosphorylation of phospholipase C-γ1 (PLC-γ1), Stat91 (one of the signal transducers and activators of transcription), and paxillin in vascular smooth muscle cells. The tyrosine kinases responsible for these phosphorylation events are unknown. Src family kinases have been shown to phosphorylate PLC-γ1 and to be activated by G protein–coupled receptors. We hypothesized that pp60c-src associates with the AT1 receptor and is activated after Ang II stimulation of smooth muscle cells. We immunoprecipitated pp60c-src from Ang II–stimulated vascular smooth muscle cells and measured pp60c-src activity by autophosphorylation and by phosphorylation of enolase. Both assays demonstrated an approximately threefold increase in pp60c-src activity within 1 minute. A similar increase in Ang II–stimulated pp60c-src activity was observed in Chinese hamster ovary cells transfected with the AT1 receptor but not in untransfected cells. These data are the first to show that pp60c-src is activated by Ang II. To determine if pp60c-src associated with the AT1 receptor, the AT1 receptor was immunoprecipitated (with two different antibodies), and Western blots were performed with two different anti-pp60c-src antibodies. No pp60c-src was detected. In addition, direct interaction between the AT1 receptor and pp60c-src could not be demonstrated by using a glutathione S-transferase (GST)-AT1 fusion protein to bind proteins from cell lysates stimulated by Ang II. In combination with recent findings that anti-pp60c-src antibodies inhibit Ang II–mediated PLC-γ1 phosphorylation, our data suggest an important role for pp60c-src in Ang II signal transduction.

Angiotensin II (Ang II) is a critical hormone in vascular homeostasis by virtue of its dual actions as a vasoconstrictor and smooth muscle cell growth factor. Of great interest is the ability of Ang II to stimulate both hyperplasia and hypertrophy of cultured vascular smooth muscle cells, implying activation of several different growth-related signal transduction events.123 These growth-related events are mediated entirely by the AT1 receptor on the basis of pharmacological effects of specific inhibitors.4 The AT1 receptor has recently been cloned,5 and its structure is typical of many G protein–coupled receptors in that it is heptahelical with a long carboxyl cytoplasmic tail. The ability of G protein–coupled receptors to stimulate cell growth in a manner similar to tyrosine kinase–coupled receptors, such as PDGF and EGF receptors, points to the importance of investigating signal transduction events in addition to those mediated by G proteins.

Several findings support the idea that activation of tyrosine kinases and phosphorylation of intracellular substrates are critical events in Ang II–stimulated vascular smooth muscle cell growth. In fact, an increase in tyrosine phosphorylation of several intracellular proteins has been identified as one of the earliest signals stimulated by Ang II.6789 More recently, specific phosphotyrosine-containing proteins have been identified as substrates for Ang II–stimulated tyrosine kinases, including PLC-γ1,10 Stat91 (from the STAT family of transcription factors),1112 and p125FAK.1314 The recent discovery that STAT proteins are phosphorylated by Ang II11 has implicated the JAK family as important mediators of Ang II signal transduction and suggests that additional similarities exist between the AT1 receptor, tyrosine kinase–coupled receptors (such as the PDGF and EGF receptors), and cytokine receptors (such as the interleukin and interferon receptors).

A pivotal role for Src family kinases has been postulated for signal transduction events mediated by both G protein–coupled receptors and cytokine receptors. In fact, Src family kinases have been demonstrated to associate with the thrombin receptor15 and cytokine receptors1617 and to be activated by ligand binding by both dephosphorylation and phosphorylation of critical tyrosine residues.18 Although the AT1 receptor has no intrinsic kinase activity, nonreceptor tyrosine kinases are capable of associating with cell surface proteins that lack intrinsic kinase activity and initiating signal transduction in a manner analogous to receptor tyrosine kinases.19 Three findings suggest that Src kinase may associate with the AT1 receptor and be important in Ang II signal transduction: (1) PLC-γ1 has been proposed to be a substrate for Src family kinases.20 By virtue of its Src homology 2 (SH2) and pleckstrin homology domains, PLC-γ1 may associate with receptors and appropriate kinases.21 Since PLC-γ1 is tyrosine-phosphorylated upon Ang II stimulation,10 it is possible that PLC-γ1 is phosphorylated by Src kinase. (2) We recently showed that the AT1 receptor is constitutively phosphorylated on serine and tyrosine residues2223 and that Src family kinases phosphorylate the carboxyl tail of the AT1 receptor in vitro.22 (3) Chen et al15 showed in fibroblasts that both Src and Fyn kinases were activated by the G protein–coupled thrombin receptor, which is similar to the AT1 receptor in many ways.2425

On the basis of these findings, we hypothesized that Src kinase associates with the AT1 receptor and is activated upon stimulation of vascular smooth muscle cells with Ang II. We found that pp60c-src was rapidly activated by Ang II binding in both cultured vascular smooth muscle cells and CHO cells transfected with the AT1 receptor. However, pp60c-src was not associated with the AT1 receptor, suggesting that pp60c-src activation occurs via interactions with other molecules involved in signal transduction.

pp60c-src Immune Complex Kinase Assay

pp60c-src Immunoprecipitates were washed three times in the buffer described above and twice in kinase reaction buffer (20 mmol/L PIPES, pH 7.0, and 10 mmol/L MnCl2). The precipitates were then suspended in the kinase reaction buffer (20 mmol/L PIPES, pH 7.0, 10 mmol/L MnCl2, and 50 μmol/L ATP) with or without 5 μg of acid-denatured (with 25 mmol/L sodium acetate, pH 3.3, 30°C, 5 minutes) rabbit muscle enolase (Sigma). The kinase reaction (final volume, 50 μL) was started by the addition of 10 μCi [γ-32P]ATP (specific activity, 3000 mCi/mmol) at 30°C and terminated after 10 minutes by the addition of SDS-PAGE sample buffer. Reactions with enolase contained an additional 30 mmol/L PIPES, pH 7.0, to neutralize the acid from the pretreatment of enolase. Samples were boiled for 5 minutes and subjected to SDS-PAGE.

Construction, Expression, and Purification of GST Fusion Protein

Polymerase chain reaction and the primers 5′-ACTGAATTCACCCTCTGTTCTACGG-3′ and 5′-TGGGAATTCGGTCGTAAGCCATTTAG-3′ with unique restriction sites were used to amplify the intracellular carboxyl tail of the AT1 receptor from pCa18b, the cDNA for the rat AT1 receptor.5 The polymerase chain reaction fragment was digested with EcoRI and ligated into EcoRI-digested pGEX-KG to give rise to pGEX-KG-AT1C (amino acids 297 to 359). The construct, which was inserted in the correct direction, expresses a fusion protein with the intracellular carboxyl tail (amino acids 297 to 359) of the AT1 receptor linked to GST (GST-AT1C [amino acids 297 to 359]). BL21 cells (Novagen) were transformed with pGEX-KG-AT1C (amino acids 297 to 359) and induced with 0.2 mmol/L isopropyl-β-thiogalactoside at 37°C for 3 hours. Cells were collected by centrifugation and lysed in buffer containing 50 mmol/L Tris, pH 8.0, 5 mmol/L EDTA, 100 mmol/L NaCl, 1 mmol/L PMSF, 10 μg/mL aprotinin, and 10 μg/mL leupeptin plus 1 mg/mL lysozyme and 0.25% sarcosyl and briefly sonicated. The lysate was cleared by centrifugation at 10 000g, and the supernatant was rocked with 50% (vol/vol) glutathione-agarose beads (Sigma) for 1 hour. The beads were collected by centrifugation at 2000g and subsequently washed three times with PBS containing 1% Triton-X, 1 mol/L NaCl, 5 mmol/L EDTA, 1 mmol/L PMSF, and 10 μg/mL leupeptin and once with PBS containing 1 mmol/L PMSF and 10 μg/mL leupeptin. GST-AT1C (amino acids 297 to 359) fusion protein was stored on beads as a 50% slurry for in vitro binding study at 4°C. The amount of fusion protein was estimated by SDS-PAGE and staining the gel with Coomassie blue.

In Vitro Binding of GST Fusion Protein to Cell Lysates

Cell lysates were mixed with 15 μg of GST-AT1 fusion protein or GST attached to glutathione-agarose beads for 3 hours at 4°C. The beads were collected by centrifugation, washed four times with cell lysis buffer, and resuspended in SDS-PAGE sample buffer.

Western Blot Analysis

Samples subjected to Western blot analysis were separated by SDS-PAGE, transferred to nitrocellulose membranes, and analyzed. After incubation in blocking solution (GIBCO BRL) overnight, membranes were incubated with primary antibodies for 1 hour at room temperature for anti-AT1 receptor antibodies, JAK-2 antibody (Santa Cruz), and anti-Src antibody SRC2 (Santa Cruz) or overnight at 4°C for anti-Src antibody mAb327. Excess primary antibody was removed by washing the membranes in PBS containing 0.03% Tween 20. The blots were incubated with appropriate secondary antibodies for 1 hour. The membranes were washed and processed for ECL (Amersham Life Science). In some experiments, membranes were reprobed after stripping in 62.5 mmol/L Tris-HCl, pH 6.8, 2% SDS, and 100 mmol/L β-mercaptoethanol for 30 minutes at 50°C.

Results

To explore the role of Src kinase in AT1 receptor signal transduction by rat vascular smooth muscle cells, we measured pp60c-src kinase activity in response to Ang II. pp60c-src was immunoprecipitated from cell lysates treated with Ang II, and pp60c-src activation was determined by autophosphorylation. pp60c-src autophosphorylation increased rapidly within 30 seconds after Ang II stimulation, and peak activation was at 1 minute (2.1-fold greater than control, Fig 1⇓). To measure the specific kinase activity of pp60c-src, phosphorylation of exogenous enolase was determined by an immune complex kinase assay. As shown in Fig 2A⇓ and 2B⇓, pp60c-src activity increased rapidly within 30 seconds in response to Ang II with a maximum 2.7-fold increase 5 minutes after Ang II stimulation. Ang II stimulation also increased the kinase activity of pp60c-src in AT1R-CHO cells with a time course similar to that observed in rat vascular smooth muscle cells. In contrast, pp60c-src was not activated by Ang II in CHO cells not transfected with the AT1 receptor (Fig 2C⇓). No difference in the amount of pp60c-src was observed in lysates from control and Ang II–stimulated cells by Western blot analysis with anti-Src antibody mAb327, indicating that the increase in pp60c-src kinase activity mediated by Ang II was not due to a change in pp60c-src protein content (data not shown).

Ang II activates pp60c-src: autophosphorylation. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared with NP-40 buffer for the pp60c-src autophosphorylation assay. The kinase reaction was performed as described in “Materials and Methods.” After SDS-PAGE, autophosphorylation was measured by densitometry in the linear range of film development. Results were confirmed by liquid scintillation counting of phosphorylated pp60c-src present in the dried gel. A, Representative autoradiogram of three experiments. B, Densitometric analysis of pp60c-src autophosphorylation. Results were normalized to control (time=0), which was arbitrarily set to 1.0.

Ang II activates pp60c-src: specific kinase activity of pp60c-src measured by enolase phosphorylation. Growth-arrested vascular smooth muscle cells or AT1R-CHO cells were treated with Ang II for the indicated times, and cell lysates were prepared with RIPA buffer for pp60c-src immune complex kinase assay using enolase as a substrate. The kinase reaction was performed as described in “Materials and Methods.” After SDS-PAGE, phosphorylation of enolase was measured by densitometry in the linear range of film development. Results were confirmed by liquid scintillation counting of phosphorylated enolase present in the dried gel. A, Representative autoradiogram of three experiments in vascular smooth muscle cells. B, Densitometric analysis of enolase phosphorylation from three independent experiments in vascular smooth muscle cells. Results were normalized to control (time=0), which was arbitrarily set to 1.0. C, Densitometric analysis of enolase phosphorylation from two independent experiments in AT1R-CHO cells and control CHO cells.

We next studied the potential interaction between pp60c-src and the AT1 receptor. We used two different antibodies in this series of experiments. The first is AT1R-COOH Ab, which was raised against a GST fusion protein expressing the intracellular carboxy tail (amino acids 306 to 359) of the AT1 receptor, and the second is AT1R-NH2 Ab, which was raised against the extracellular amino terminus (amino acids 15 to 24). Western blot analysis of rat vascular smooth muscle cell membranes with the AT1R-COOH Ab demonstrated proteins with molecular masses of 48 and 60 kD. These weights are identical to those previously reported for the AT1 receptor22 and likely represent nascent and mature glycosylated receptors, respectively.28 AT1R-NH2 Ab detected a protein with a molecular mass of 48 kD on a similar Western blot (Fig 3⇓). Because the peptide (amino acids 15 to 24) used as an antigen for AT1R-NH2 Ab is close to a potential glycosylation site (amino acid 4), this antibody may not recognize the mature glycosylated 60-kD AT1 receptor, explaining the difference shown in Fig 3⇓. Immunoprecipitation of the AT1 receptor from rat vascular smooth muscle cells with the AT1R-COOH Ab identified a protein with a molecular mass of 60 kD (Fig 4A⇓). However, Western blot analysis of the AT1R-COOH Ab immunoprecipitates with the AT1R-COOH Ab failed to identify the 48-kD AT1 receptor, because IgG heavy chain (molecular mass, 46 to 48 kD) was recognized by the secondary antibody. Since the AT1R-COOH Ab was not affinity-purified, we confirmed that an antibody against GST protein did not precipitate the protein that reacted with the AT1R-COOH Ab (data not shown). We confirmed that several proteins were coimmunoprecipitated with the AT1 receptor by silver staining (data not shown). The AT1 receptor immunoprecipitates were analyzed on Western blots for specific proteins. One of the coimmunoprecipitated proteins was identified as JAK-2, a tyrosine kinase (Fig 4B⇓) as reported previously.12 However, pp60c-src was not detected in the AT1 receptor immunoprecipitates (Fig 4C⇓). Thus, the failure to detect pp60c-src was unlikely an artifact due to the conditions used for immunoprecipitation.

Western blot (WB) analyses of rat vascular smooth muscle cell membranes with two different AT1 receptor antibodies. Rat vascular smooth muscle cell membranes were isolated and prepared for WB analysis as described in “Materials and Methods.” Membrane proteins were electrophoresed, and WB analysis was performed with the indicated antibodies (AT1R-COOH and AT1R-NH2).

Ang II does not stimulate binding of pp60c-src to the AT1 receptor immunoprecipitated with AT1R-COOH antibody. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared for immunoprecipitation of the AT1 receptor with the AT1R-COOH antibody. The immunoprecipitated proteins were electrophoresed, and Western blot (WB) analysis was performed with the indicated antibodies. A, Immunoprecipitation (IP) with AT1R-COOH Ab and WB analysis with AT1R-COOH Ab. Arrowhead indicates the 60-kD immunoreactive protein that is the AT1 receptor. B, IP with AT1R-COOH Ab and WB analysis with JAK-2 antibody. Arrow indicates the 120-kD coimmunoprecipitated JAK-2. C, IP with AT1R-COOH Ab and WB analysis with mAb327 against pp60c-src (Src). Results are representative of three experiments.

It is possible that the AT1R-COOH Ab may interfere with pp60c-src binding to the AT1 receptor, since both AT1R-COOH Ab and pp60c-src could interact with the carboxyl tail.22 Therefore, we used two additional methods to confirm the lack of association between the AT1 receptor and pp60c-src. First, we used an antibody against the extracellular NH2 terminus, AT1R-NH2 Ab, to immunoprecipitate the AT1 receptor. As shown in Fig 5A⇓, pp60c-src was not detected when the AT1 receptor immunoprecipitates were analyzed on Western blots with an anti-pp60c-src antibody mAb327, although the AT1 receptor was immunoprecipitated (Fig 5B⇓). In addition, pp60c-src was not detected in AT1 receptor immunoprecipitates when another anti-Src antibody, SRC2, was used (data not shown). No change in pp60c-src immunoreactive protein was observed in the supernatants after immunoprecipitation of the AT1 receptor (Fig 5A⇓). Second, to examine whether pp60c-src binds directly to the carboxyl tail of the AT1 receptor in vitro, a fusion protein, GST-AT1C (amino acids 297 to 359), was expressed in bacteria, purified on the glutathione-agarose beads, and incubated with vascular smooth muscle cell lysates. Proteins that associated with the GST-AT1C (amino acids 297 to 359) fusion protein on beads were subjected to Western blot analysis with anti-pp60c-src antibody mAb327. Of interest, the presence of a protein kinase in these precipitates could be readily demonstrated. As shown in Fig 6B⇓, addition of [γ-32P]ATP to these precipitates resulted in significant phosphorylation of the GST-AT1C (amino acids 297 to 359) fusion protein. There appeared to be little regulation of this kinase activity by Ang II under the conditions used. As shown in Fig 6A⇓, pp60c-src was not detected in the coprecipitates with the GST-AT1C (amino acids 297 to 359) fusion protein as assayed by Western blot with anti-pp60c-src antibody mAb327. In summary, these results indicate that pp60c-src does not directly associate with the AT1 receptor.

Ang II does not stimulate binding of pp60c-src to the AT1 receptor immunoprecipitated with AT1R-NH2 antibody. Growth-arrested vascular smooth muscle cells were treated with Ang II for the indicated times, and cell lysates were prepared for immunoprecipitation (IP) of the AT1 receptor with the AT1R-NH2 Ab. A, The AT1 receptor was immunoprecipitated with AT1R-NH2 Ab. The immunoprecipitated proteins and the supernatants remaining after IP were electrophoresed, and Western blot (WB) analysis was performed with mAb327 against pp60c-src (Src). B, The membrane was stripped and reprobed with AT1R-NH2 Ab. Arrows indicate the 48-kD AT1 receptor immediately below the rabbit IgG. Results are representative of three experiments.

pp60c-src does not directly bind to the GST-AT1 fusion protein. Growth-arrested vascular smooth muscle cells were left unstimulated (−) or stimulated for 5 minutes with 100 nmol/L Ang II (+). Cell lysates were prepared and incubated with GST alone (lanes 1 and 2) or GST-AT1C (amino acids 297 to 359) fusion protein (lanes 3 and 4) for 3 hours at 4°C. A, The coprecipitated protein and the supernatants remaining after binding of GST-AT1C (amino acids 297 to 359) fusion protein were resolved by SDS-PAGE, and Western blot analysis was performed with mAb327 against pp60c-src (Src). No pp60c-src was detected in the GST-AT1C (amino acids 297 to 359) precipitates with (+) or without (−) Ang II stimulation. pp60c-src was readily detected in the supernatant (arrow). B, The coprecipitated proteins were subjected to in vitro kinase assay as described in “Materials and Methods” and resolved by SDS-PAGE. “Mock” indicates that GST-AT1C (amino acids 297 to 359) fusion protein was subjected to in vitro kinase assay without the addition of cell lysate.

Discussion

The most important finding of the present study is the demonstration that Ang II activates pp60c-src in vascular smooth muscle cells, and this is the first study to demonstrate it. Since the AT1 receptor was cloned, there has been a rapid increase in our understanding of the signal transduction mechanisms responsible for the effects of Ang II. Perhaps most impressive has been the identification of a number of serine/threonine kinases activated by Ang II binding that initiate signal transduction. These include Raf kinase, mitogen-activated protein kinase, protein kinase C, calcium calmodulin–dependent kinase, and S6 kinase.29 Although the AT1 receptor is a typical G protein–coupled receptor in that it lacks tyrosine kinase activity, many proteins are rapidly tyrosine-phosphorylated when Ang II binds to the AT1 receptor. This suggests that a receptor-associated tyrosine kinase(s) may be involved in Ang II signaling. Because the AT1 receptor is itself tyrosine-phosphorylated,23 binding of SH2-containing proteins, such as pp60c-src, may be a possible mechanism for activation of the receptor-associated kinase. However, the present study suggests that pp60c-src does not bind to the AT1 receptor despite being rapidly activated in cells treated with Ang II. Despite our inability to show a direct association between the AT1 receptor and pp60c-src, it is possible that such an association occurs in vivo. For example, the association may be transient and impossible to detect by immunoprecipitation, or a low-affinity interaction, mediated by other cell proteins, may occur. The failure of the AT1-GST fusion protein to bind pp60c-src may be explained by improper folding due to the presence of GST or a requirement for other intracellular loops (eg, the third intracellular loop) to maintain the interaction between the AT1 receptor and pp60c-src.

A large body of evidence suggests that Src kinase is important in Ang II–stimulated events in vascular smooth muscle cells. First, it has been demonstrated that both p125FAK and paxillin are tyrosine-phosphorylated rapidly in response to Ang II.1314 p125FAK has been identified to be important in paxillin phosphorylation.30 Because p125FAK is itself activated by pp60c-src-mediated phosphorylation of tyrosines 576 and 577,31 it appears plausible that Src kinase is involved in Ang II–mediated p125FAK and paxillin phosphorylation. Second, α-thrombin, which stimulates many of the same events as Ang II in vascular smooth muscle cells,12425 has been found to activate Src family kinases in fibroblasts with a time course that is very similar to that shown here for Ang II.15 In addition, Src kinase has been shown to be involved in signal transduction mediated by other G protein–coupled receptors, including the receptors for lysophosphatidic acid,32 endothelin,33 and platelet-activating factor receptors.34 Third, we showed previously that the AT1 receptor was rapidly tyrosine-phosphorylated by Src family kinases in vitro.22 Fourth, we have shown that PLC-γ1 is rapidly tyrosine-phosphorylated in response to Ang II, with peak phosphorylation at 1 minute and that the phosphorylation and activation of PLC-γ1 are decreased by the tyrosine kinase inhibitors genistein and tyrphostin.10 Very recent data from our laboratory show that electroporation of an antibody against pp60c-src specifically inhibits PLC-γ1 phosphorylation and activity.35 Thus, it appears that pp60c-src is responsible for tyrosine phosphorylation of PLC-γ1 in response to Ang II in vascular smooth muscle cells. Finally, we have recently shown that the receptor-associated kinases of the JAK family bind to the AT1 receptor and phosphorylate the STAT family of transcription factors.36 Of interest, Stat3-related protein has recently been found to be activated by the Src oncogene tyrosine kinase.37 Thus, it is likely that Src kinase plays an important role in Ang II signal transduction.

The present findings support the concept that the AT1 receptor acts as a cytokine-like receptor. Early signal transduction events mediated by cytokine receptors include activation of receptor-associated kinases, including JAK, TYK, and the Src family kinases.16173839 The fact that the receptor-associated kinases of the JAK family bind to the AT1 receptor and phosphorylate the STAT family of transcription factors36 suggests that the long-term effects of Ang II on gene expression may resemble those of tyrosine kinase–coupled receptors, such as the PDGF receptor, and classic cytokine receptors, such as the interleukin and interferon receptors.3940 In the present study, this similarity is extended to the Src family kinases, specifically pp60c-src. In several cytokine and tyrosine kinase–coupled receptors, pp60c-src directly associates with the receptors.16 However, this mechanism of interaction does not pertain to the AT1 receptor on the basis of the present study. If pp60c-src does not bind to the AT1 receptor, how may it be localized to the receptor and be activated? One potential mechanism is suggested by the recent findings that the β-adrenergic receptor kinase associates with the β-adrenergic receptor by virtue of Gβγ subunits interacting with the pleckstrin homology domain of the β-adrenergic receptor kinase.41 Interaction between Gβγ subunits, their associated kinases, and kinase substrates may provide the signaling complex that activates and binds pp60c-src. Thus, future studies should identify the mechanisms by which Src kinase is activated upon binding of Ang II to the AT1 receptor.

Acknowledgments

This study was supported by grants from the National Institutes of Health to Drs Berk, Bernstein, and Marrero and a grant from the Japan Heart Foundation to Dr M. Ishida. Drs Berk and Bernstein are Established Investigators of the American Heart Association. We thank members of the Berk laboratory, especially Drs M. Kusuhara, J. L. Duff, and T. E. Peterson, for their help.